Polymeric microparticles containing agents for imaging

ABSTRACT

Compositions, methods for preparing and methods of using contrast agent-filled polymeric microparticles for imaging are disclosed. In a preferred embodiment, air-encapsulating microparticles are formed by ionotropically gelling synthetic polyelectrolytes such as poly(carboxylatophenoxy)phosphazene, poly(acrylic acid), poly(methacrylic acid) and methacrylic acid copolymers (Eudragit&#39;s) by contact with multivalent ions such as calcium ions. In the preferred embodiment, the average size of the microparticles is less than seven μm so that they are suitable for injection intravenously. The polymeric microparticles are stable to imaging and display high echogenicity, both in vitro and in vivo. Due to their in vivo stability their potential application is extended beyond vascular imaging to liver and renal diseases, fallopian tube diseases, detecting and characterizing tumor masses and tissues, and measuring peripheral blood velocity. The microparticles can optionally be linked with ligands that minimize tissue adhesion or that target the microparticles to specific regions.

BACKGROUND OF THE INVENTION

This is a continuation-in-part of U.S. Ser. No. 08/214,860 filed Mar.18, 1994, which is a continuation-in-part of U.S. Ser. No. 08/182,216filed Jan. 14, 1994, by Smadar Cohen, Alexander K. Andrianov, MargaretWheatley, Harry R. Allcock and Robert S. Langer now U.S. Pat. No.5,487,390, which is a continuation in part of U.S. Ser. No. 07/880,248,filed on May 8, 1992, by Smadar Cohen, Carmen Bano, Karyn Visscher,Marie Chow, Harry Allcock, and Robert Langer, now U.S. Pat. No.5,308,701, which is a divisional application of U.S. Ser. No.07/593,684, filed on Oct. 5, 1990, by Smadar Cohen, Carmen Bano, KarynVisscher, Marie Chow, Harry Allcock, and Robert Langer, now U.S. Pat.No. 5,149,543.

This invention is in the area of polymeric delivery systems, and inparticular is polymeric microparticles that encapsulate gas and methodsfor their preparation and use.

Diagnostic ultrasound is a powerful, non-invasive tool that can be usedto obtain information on the internal organs of the body. The advent ofgrey scale imaging and color Doppler have greatly advanced the scope andresolution of the technique. Although techniques for carrying outdiagnostic ultrasound have improved significantly, there is still a needto enhance the resolution of the imaging for: (i) cardiac, solid organ,and vascular anatomic conduits (for example, the imaging of macrophageactivity); (ii) solid organ perfusion; and (iii) Doppler signals ofblood velocity and flow direction during real-time imaging.

Traditional, simple ultrasonic echograms reveal blood vessel walls andother echo-producing structures. However, since echoes from bloodnormally are not recorded, identifying which echoes are from which bloodvessels is usually difficult. For example, echoes from the far wall ofone blood vessel can be confused with the near wall of an adjacent bloodvessel, and vice versa.

Ultrasonic contrast agents can be used to increase the amount ofultrasound reflected back to a detector. Ultrasonic contrast mediumsfill the entire intraluminal space with echoes and readily permitidentification of the correct pair of echoes corresponding to the wallsof a particular blood vessel.

Ultrasonic contrast agents are primarily used in high-flow systems inwhich the contrast enhancement can be quickly evanescent. Forechocardiography, a full display of bubble agents, ranging in size fromtwo μm to 12 μm, and persisting from two or three to 30 seconds, hasbeen used. For other applications, such as neurosonography,hysterosalpingography, and diagnostic procedures on solid organs, theagent must have a lifetime of more than a few circulation times andconcentrate in organ systems other than the vascular tree into which itis injected. It must also be small enough to pass through the pulmonarycapillary bed (less than eight microns).

Aqueous suspensions of air microbubbles are the preferred echo contrastagents due to the large differences in acoustic impedance between airand the surrounding aqueous medium. After injection into the bloodstream, the air bubbles should survive at least for the duration ofexaminations. The bubbles should be injectable intravenously and smallenough to pass through the capillaries of the lungs.

The simplest suspension of air bubbles has been obtained by handagitation of 70% dextrose or sorbitol solutions. However, this methodproduces large bubbles with an average diameter of greater than 15 μmthat exhibit a very limited in vitro stability (less than 1 minute).Feinstein, S. B., et al., J. Am. Coll. Cardiol., Vol. 3, pp. 14-20(1984); Keller, M. W., et al., J. Ultrasound Med., Vol. 5, pp. 493-498(1986). Smaller bubbles (usually approximately five μm in diameter) havebeen obtained by sonicating solutions of 50% or 70% dextrose orRenografin-76 (diatrizoate meglumin 66%) but their in vitro persistencestill seldom exceeds a few minutes (Feinstein, J. Am. Coll. Cardiol. 11,59-65 (1988), and Keller, (1988)), and their in vivo persistence only afew seconds. This short lifetime may be appropriate for someapplications in cardiology but may not be sufficient for organ imaging.

Air-filled particles with a polymeric shell should exhibit a longerpersistence after injection than a nonpolymeric microbubble, and may besuitable not only for cardiology but also for organ and peripheral veinimaging. A variety of natural and synthetic polymers have been used toencapsulate imaging contrast agents, such as air. Research efforts inthis area have to date primarily focused on agarose and alginate as theencapsulating polymers.

Agarose gel microbeads can be formed by emulsifying agarose-parafilm oilmixtures or through the use of teflon molds. In both cases,temperature-mediated gelation of agarose requires temperature elevationsthat render difficult the encapsulation gaseous imaging contrast agents.

Alginate, on the other hand, can be ionically cross-linked with divalentcations, in water, at room temperature, to form a hydrogel matrix, asdescribed by Wheatley, et al., Biomaterials 11, 713-718 (1990) and Kwok,K. K., et al., Pharm. Res., Vol. 8(3) pp. 341-344 (1991). Wheatley, etal., produced ionically crosslinked microcapsules less that ten micronsin diameter, where were formed of alginate, encapsulating air, for usein diagnostic ultrasound. Kwok produced microparticles in the range of 5to 15 μm by spraying a sodium alginate solution from an air-atomizingdevice into a calcium chloride solution to effect crosslinking, and thenfurther crosslinking the resulting microcapsules with poly-L-lysine.

Schneider, et al., Invest. Radiol., Vol. 27, pp. 134-139 (1992)described three micron, air-filled polymeric particles. These particleswere stable in plasma and under applied pressure. However, at 2.5 MHz,their echogenicity was low. Another drawback of these particles was thatorganic solvents (tetrahydrofuran and cyclohexane) were used to preparethe particles. Organic solvents can be difficult to remove from themicrobubble and may cause a health risk to the patient.

Another type of microbubble suspension has been obtained from sonicatedalbumin. Feinstein, et al., J. Am. Coll. Cardiol., Vol. 11, pp. 59-65(1988). Feinstein describes the preparation of microbubbles that areappropriately sized for transpulmonary passage with excellent stabilityin vitro. However, these microbubbles are short-lived in vivo (T1/2 =few seconds, which is approximately equal to one circulation pass)because of their instability under pressure (Gottlieb, S., et al., J.Am. Soc. Echo, Vol. 3, pp. 328 (1990), Abstract; Shapiro, J. R., et al.,J. Am. Coll. Cardiol., Vol. 16, pp. 1603-1607 (1990)).

Gelatin-encapsulated air bubbles have been described by Carroll, et al.(Carroll, B. A., et al., Invest. Radiol., Vol. 15, pp. 260-266 (1980);and Carroll, B. A., et al., Radiology, Vol. 143, pp. 747-750 (1982)),but due to their large sizes (12 and 80 μm) they would likely not passthrough pulmonary capillaries. Gelatin-encapsulated microbubbles havealso been described in PCT/US80/00502 by Rasor Associates, Inc. Theseare formed by "coalescing" the gelatin.

Microbubbles stabilized by microcrystals of galactose (SHU 454 and SHU508) have also been reported by Fritzsch, T., et al., Invest. Radiol.Vol. 23 (Suppl 1), pp. 302-305 (1988); Fritzsch, T., et al., Invest.Radiol., Vol. 25 (Suppl 1), 160-161 (1990). The microbubbles last up to15 minutes in vitro but less than 20 seconds in vivo (Rovai, D., et al.,J. Am. Coll. Cardiol., Vol. 10, pp. 125-134 (1987); Smith, M., et al.,J. Am. Coll. Cardiol., Vol. 13, pp. 1622-1628 (1989).

A disadvantage of using natural polymers is that their biocompatibilityis variable, and, due to impurities in the preparation extracts, it isdifficult to reproduce some properties of the polymer. Syntheticpolymers are preferable because they are reproducible and theirproperties can be tailored to specific needs, includingbiodegradability.

Synthetic polymers are used increasingly in medical science since theycan incorporate specific properties such as strength, hydrogelcharacteristics, permeability and biocompatability, particularly infields like cell encapsulation and drug delivery, where such propertiesare often prerequisites. However, typical methods for the fabrication ofsynthetic polymers into matrices or drug delivery particles involveheat, which makes encapsulating gaseous imaging contrast agentsparticularly difficult, or organic solvents, which may be injurious tothe health of the patient.

European Patent Application No. 91810366.4 by Sintetica S. A. (0 458 745A1) discloses air or gas microballoons bounded by an interfaciallydeposited polymer membrane that can be dispersed in an aqueous carrierfor injection into a host animal or for oral, rectal, or urethraladministration, for therapeutic or diagnostic purposes. Themicroballoons are prepared by the steps of: emulsifying a hydrophobicorganic phase into a water phase to obtain an oil-in-water emulsion;adding to the emulsion at least one polymer in a volatile organicsolvent that is insoluble in the water phase; evaporating the volatilesolvent so that the polymer deposits by interfacial precipitation aroundthe hydrophobic phase in the water suspension; and subjecting thesuspension to reduced pressure to remove the hydrophobic phase and thewater phase in a manner that replaces air or gas with the hydrophobicphase. There are two major disadvantages of this process. First, onlypolymers that have very specific solubility profiles can be used toprepare the microbubbles, i.e., they must be "interfacially depositable"on a hydrophobic phase in an aqueous medium, and soluble in a volatileorganic solvent that is water-insoluble. Second, the process requiresthe use of organic solvents, which may be hard to completely remove fromthe microbubble and which may be injurious to the patient's health.

It would be useful to have a method to encapsulate imaging contrastagents with biodegradable or nonbiodegradable synthetic polymers thatcan be accomplished without the use of elevated temperatures or organicsolvents.

Another disadvantage of current microbubble technology is the tendencyof the microbubble to adhere to tissues, and the inability toeffectively target the microbubbles to specific regions of interest inthe body, for example, a solid tumor site or disperse tumor cells. Itwould be desirable to have a polymeric microbubble that has a surfacethat minimizes tissue adhesion, or that can be designed to target tospecific regions in the body.

It is therefore an object of the present invention to providemicroparticles made from synthetic polymers containing an imaging agent.

It is another object of the present invention to provide microparticlescontaining an imaging agent that can persist for more than a fewcirculation times.

It is still another object of the present invention to providemicroparticles containing imaging agents that can be prepared withoutthe use of heat or organic solvents.

It is a further object of the present invention to provide methods forpreparing these microparticles containing imaging agents.

It is still a further object of the present invention to providemicroparticles containing imaging agents that do not adhere to tissues.

It is yet a further object of the invention to provide microparticlescontaining imaging agents that are targeted to specific regions of thebody.

SUMMARY OF THE INVENTION

Ionically crosslinked synthetic polymeric microparticles containingimaging agents, and methods for their preparation and use, aredisclosed. The polymeric microparticles are useful in diagnosticultrasound imaging, magnetic resonance imaging (MRI), fluroscopy, x-ray,and computerized tomography, and can be prepared in micron and submicronsizes that are injectable and that are capable of passing through thepulmonary capillary bed.

The microparticles are prepared by crosslinking a water-solublesynthetic polymer that contains charged side chains with multivalentions of the opposite charge. The product, typically a hydrogel, isoptionally further stabilized by exposing the product to multivalentpolyions, preferably in the form of an ionic polymer, of the same chargeas those used to form the hydrogel.

In the preferred embodiment, hydrolytically stablepoly(organophosphazenes) such as poly(carboxylatophenoxy)phosphazene andits copolymers, poly(acrylic acid), poly(methacrylic acid) ormethacrylic acid copolymers, that contain carboxylic acid, sulfonic acidor hydroxyl substituent groups, are crosslinked with divalent ortrivalent pharmaceutically acceptable cations such as zinc, calcium,bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, chromium,or cadmium, most preferably zinc.

The microparticles can be targeted to specific regions of the body bycovalently binding to the polymer a targeting molecule. The targetingmolecule can be, for example, a protein or peptide (such as a hormone,antibody or antibody fragment such as the Fab or Fab₂ antibodyfragments, or a specific cell surface receptor ligand), lipid,polysaccharide, nucleic acid, carbohydrate, a combination thereof, orother molecule, including a synthetic molecule, that identifies andlocalizes at the target material.

The microparticles can also be designed to minimize tissue adhesion bycovalently binding a poly(alkylene glycol) moiety to the surface of themicroparticle. The surface poly(alkylene glycol) moieties have a highaffinity for water that reduces protein adsorption onto the surface ofthe particle. The recognition and uptake of the microparticle by thereticulo-endothelial system (RES) is therefore reduced.

In one embodiment, the terminal hydroxyl group of the poly(alkyleneglycol) can be used to covalently attach biologically active molecules,or molecules affecting the charge, lipophilicity or hydrophilicity ofthe particle, onto the surface of the microparticle. The biologicallyactive molecule can be a protein, carbohydrate or polysaccharide,nucleic acid, lipid, a combination thereof, or a synthetic molecule,including organic and inorganic materials.

In a preferred embodiment, the microparticles are prepared by sonicatingsolutions of synthetic polymer, typically using ultrasonic frequenciesof between 5,000 and 30,000 Hz, to produce a highly aerated gassedsolution, and spraying the polymer solution into a solution ofmultivalent ions. Microparticles produced by this method are typicallysmaller than seven microns. In a preferred embodiment, themicroparticles have a diameter in the range of between approximately oneand seven microns.

DETAILED DESCRIPTION OF THE INVENTION

Synthetic polymers with ionically crosslinkable groups are crosslinkedin solutions of ions of the opposite charge containing imaging agents toencapsulate the imaging contrast agents. The resulting product is arelatively homogenous population of hydrogel microparticles containingone or more imaging agents. As used herein, a "microparticle" refers toa hydrogel particle having one or more imaging agents entrapped therein.In one embodiment, the outer layer is crosslinked with a multivalentpolyion and the core material is a liquid of the same or differentmaterial as the hydrogel.

Ionic crosslinking occurs in solutions of anionic polyelectrolytes andcations, or cationic polyelectrolytes and anions, due to strongelectrostatic forces surrounding the polymeric chains. The formation andproperties of polymers crosslinked via polyvalent ions depend on theproperties, concentrations, and distribution of the ions and thepolymer. Polymer chains crosslink via cations or anions by formingcomplexes liganded with more than one polymer group, creatingintramolecular and/or intermolecular crosslinks (C. Allain and L.Salome, Macromolecules, Vol. 23, pp. 981-987 (1990).

In the preferred embodiment, sterilized air is encapsulated withinhydrogel microparticles. These can be subsequently further crosslinkedwith charged polymers to form an outer permeable membrane and can beconverted into microcapsules by liquefying the core hydrogel. Otherembodiments include imaging agents useful in x-ray imaging, fluoroscopy,magnetic resonance imaging, and computerized tomography.

An important element of the method and the resulting microparticulatesis that the method uses water soluble, synthetic polymers. The methodfor making the microparticles therefore does not require the use ofnon-water-miscible organic solvents, is highly reproducible and requiresfew processing steps. Synthetic polymers are selected that arebiocompatible, and are at least partially soluble in aqueous solutions,or which form a dispersion in an aqueous solution. In a preferredembodiment, the synthetic polymer is biodegradable over a short periodof time, usually a few days to one week. The rate of hydrolysis of thepolymer can typically be manipulated so that it can be processed andremain intact for a desired period of time.

In a preferred embodiment, microparticles exhibit an in vivo lifetime offrom approximately thirty seconds to thirty minutes or more, or at leastenough time to be delivered to the region of interest and for theultrasound operator to carry out the diagnostic tests.

I. Selection, Synthesis, Crosslinking, and Modification of Water-SolublePolyelectrolyte Polymers

A number of polymers can be used to form the cross-linked hydrogel. Ingeneral, polymers that are suitable are those that have charged sidegroups and are at least partially soluble in aqueous solutions, such aswater, buffered salt solutions, or aqueous alcohol solutions, or amonovalent ionic salt thereof. As used herein, "partially soluble"refers to a polymer that is soluble to the extent of at least 0.001percent by weight of aqueous solution, and preferably, soluble to anextent of at least 0.01 percent by weight.

Examples of polymers with acidic side groups that can be reacted withcations include those with carboxylic acid, sulfonic acid, sulfamicacid, phosphoric acid, phosphonic acid, hydroxyl or thiol groups withacidic hydrogens (for example, halogenated, preferably fluorinated,alcohols), boric acid, or any other moiety that will react with a cationto form a conjugate base. Examples include poly(organophosphazenes) withacidic substituent groups, poly(acrylic acids), poly(methacrylic acids),copolymers of acrylic acid and methacrylic acid, copolymers of vinylacetate with acidic monomers, sulfonated polymers, such as sulfonatedpolystyrene, and copolymers formed by reacting acrylic or methacrylicacid and vinyl ether monomers or polymers.

Examples of polymers with basic side groups that can be reacted withanions include those that have amino, imino, mono- or di-(alkyl oraryl)amino, heterocyclic nitrogen, or quaternary amino groups, andspecifically include poly(vinyl amines), poly(vinyl pyridine),poly(vinyl imidazole), poly(vinyl pyrrolidone) and some amino orsubstituted polyphosphazenes. The ammonium quaternary salt of thepolymers can also be formed from the backbone nitrogens or pendant iminogroups.

A. Synthesis and Selection of Polymers

1. Polyphosphazenes

Polyphosphazenes are polymers with backbones consisting of alternatingphosphorus and nitrogen, separated by alternating single and doublebonds. Each phosphorous atom is covalently bonded to two pendant groups("R"). The substituent ("R") can be any of a wide variety of moietiesthat can vary within the polymer, including but not limited toaliphatic, aryl, aralkyl, alkaryl, carboxylic acid, heteroaromatic,carbohydrates, including glucose, heteroalkyl, halogen,(aliphatic)amino- including alkylamino-, heteroaralkyl,di(aliphatic)amino-including dialkylamino-, arylamino-, diarylamino-,alkylarylamino-, -oxyaryl including but not limited to -oxyphenylCO₂ H,-oxyphenylSO₃ H, -oxyphenylhydroxyl and -oxyphenylPO₃ H; -oxyaliphaticincluding -oxyalkyl, -oxy(aliphatic)CO₂ H, -oxy(aliphatic)SO₃ H,-oxy(aliphatic)PO₃ H, and -oxy(aliphatic)hydroxyl, including-oxy(alkyl)hydroxyl; -oxyalkaryl, -oxyaralkyl, -thioaryl, -thioaliphaticincluding -thioalkyl, -thioalkaryl, -thioaralkyl, -NHC(O)O-(aryl oraliphatic), --O--[(CH₂)_(x) O]_(y) --CH₂)_(x) NH₂, --O--[(CH₂)_(x)O]_(y) CH₂)_(x) NH(CH2)_(x) SO₃ H, and --O--[(CH₂)_(x) O]_(y) -(aryl oraliphatic), wherein x is 1-8 and y is an integer of 1 to 20. The groupscan be bonded to the phosphorous atom through, for example, an oxygen,sulfur, nitrogen, or carbon atom. The polymers can be designed to behydrophobic, amphophilic, or hydrophilic; water-stable orwater-erodible; crystalline or amorphous; or bioinert or bioactive.

The term amino acid, as used herein, refers to both natural andsynthetic amino acids, and includes, but is not limited to alanyl,valinyl, leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryptophanyl,methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl,asparaginyl, glutaminyl, aspartoyl, glutaoyl, lysinyl, argininyl, andhistidinyl.

The term amino acid ester refers to the aliphatic, aryl orheteroaromatic carboxylic acid ester of a natural or synthetic aminoacid.

The term alkyl, as used herein, refers to a saturated straight,branched, or cyclic hydrocarbon, or a combination thereof, typically ofC₁ to C₂₀, and specifically includes methyl, ethyl, propyl, isopropyl,butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl,hexyl, isohexyl, cyclohexyl, 3-methylpentyl, 2,2-dimethylbutyl,2,3-dimethylbutyl, heptyl, octyl, nonyl, and decyl.

The term (alkyl or dialkyl)amino refers to an amino group that has oneor two alkyl substituents, respectively.

The terms alkenyl and alkynyl, as used herein, refers to a C₂ to C₂₀straight or branched hydrocarbon with at least one double or triplebond, respectively.

The term aryl, as used herein, refers to phenyl or substituted phenyl,wherein the substituent is halo, alkyl, alkoxy, alkylthio, haloalkyl,hydroxyalkyl, alkoxyalkyl, methylenedioxy, cyano, C(O)(lower alkyl),--CO₂ H, --SO₃ H, --PO₃ H, --CO₂ alkyl, amide, amino, alkylamino anddialkylamino, and wherein the aryl group can have up to threesubstituents.

The term aliphatic refers to hydrocarbon, typically of C₁ to C₂₀ , thatcan contain one or a combination of alkyl, alkenyl, or alkynyl moieties,and which can be straight, branched, or cyclic, or a combinationthereof.

The term halo, as used herein, includes fluoro, chloro, bromo, and iodo.

The term aralkyl refers to an aryl group with an alkyl substituent.

The term alkaryl refers to an alkyl group that has an aryl substituent,including benzyl, substituted benzyl, phenethyl or substitutedphenethyl, wherein the substituents are as defined above for arylgroups.

The term heteroaryl or heteroaromatic, as used herein, refers to anaromatic moiety that includes at least one sulfur, oxygen, or nitrogenin the aromatic ring, and that can be optionally substituted asdescribed above for aryl groups. Nonlimiting examples are furyl,pyridyl, pyrimidyl, thienyl, isothiazolyl, imidazolyl, tetrazolyl,pyrazinyl, benzofuranyl, benzothiophenyl, quinolyl, isoquinolyl,benzothienyl, isobenzofuryl, pyrazolyl, indolyl, isoindolyl,benzimidazolyl, purinyl, carbozolyl, oxazolyl, thiazolyl, isothiazolyl,1,2,4-thiadiazolyl, isooxazolyl, pyrrolyl, pyrazolyl, quinazolinyl,pyridazinyl, pyrazinyl, cinnolinyl, phthalazinyl, quinoxalinyl,xanthinyl, hypoxanthinyl, pteridinyl, 5-azacytidinyl, 5-azauracilyl,triazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl, andpyrazolopyrimidinyl.

The term heteroalkyl, as used herein, refers to a alkyl group thatincludes a heteroatom such as oxygen, sulfur, or nitrogen (with valencecompleted by hydrogen or oxygen) in the carbon chain or terminating thecarbon chain.

In one embodiment, the poly(organophosphazene) contains (i) ionized orionizable pendant groups, and (ii) pendant groups that are susceptibleto hydrolysis under the conditions of use, to impart biodegradability tothe polymer. Suitable hydrolyzable groups include, for example,chlorine, amino acid, amino acid ester, imidazole, glycerol, andglucosyl.

The degree of hydrolytic degradability of the polymer will be a functionof the percentage of pendant groups susceptible to hydrolysis and therate of hydrolysis of the hydrolyzable groups. The hydrolyzable groupsare believed to be replaced by hydroxyl groups in aqueous environmentsto provide P--OH bonds that impart hydrolytic instability to thepolymer.

While the acidic or basic groups are usually on nonhydrolyzable pendantgroups, they can alternatively, or in combination, also be positioned onhydrolyzable groups.

In a typical embodiment, a portion, generally 10% or less, of the sidechain groups (the R groups in formula 1), are susceptible to hydrolysis.

Specific examples of hydrolyzable side chains are unsubstituted andsubstituted imidizoles and amino acid esters in which the group isbonded to the phosphorous atom through an amino linkage (polyphosphazenepolymers in which both R groups are attached in this manner are known aspolyaminophosphazenes).

In polyimidazolephosphazenes, some of the "R" groups on thepolyphosphazene backbone are imidazole rings, attached to phosphorous inthe backbone through a ring nitrogen atom. Other "R" groups can beorganic residues that do not participate in hydrolysis, such as methylphenoxy groups or other groups shown in Allcock, at al., Macromolecule10:824-830 (1977), hereby incorporated by reference.

Specific examples of R groups that are not capable of hydrolysis arealkyl, aralkyl, or aryl group having 20 carbon atoms or less (morepreferably 12 carbon atoms or less); or a heteroalkyl or heteroarylgroup having 20 or less carbons and heteroatoms (more preferably 12 orless carbon or heteroatoms). If the alkyl chain is too long, the polymerwill be totally insoluble in water. The groups can be bonded to thephosphorous atom through e.g., an oxygen, sulfur, nitrogen, or carbonatom.

In general, when the polyphosphazene has more than one type of pendantgroup, the groups will vary randomly throughout the polymer, and thepolyphosphazene is thus a random copolymer. Phosphorous can be bound totwo like groups, or two different groups. Polyphosphazenes with two ormore types of pendant groups can be produced by reactingPoly(dichlorophosphazene) with the desired nucleophile or nucleophilesin a desired ratio. The resulting ratio of pendant groups in thepolyphosphazene will be determined by a number of factors, including theratio of starting materials used to produce the polymer, the temperatureat which the nucleophilic substitution reaction is carried out, and thesolvent system used. While it is very difficult to determine the exactsubstitution pattern of the groups in the resulting polymer, the ratioof groups in the polymer can be easily determined by one skilled in theart.

It should be understood that certain groups, such as heteroaromaticgroups other than imidazole, hydrolyze at an extremely slow rate underneutral aqueous conditions, such as that found in the blood, andtherefore are typically considered nonhydrolyzable groups for purposesherein. However, under certain conditions, for example, low pH, asfound, for example, in the stomach, the rate of hydrolysis of normallynonhydrolyzable groups (such as heteroaromatics other than imidazole)can increase to the point that the biodegradation properties of thepolymer can be affected. One of ordinary skill in the art using wellknown techniques can easily determine whether pendant groups hydrolyzeat a significant rate under the conditions of use. One of ordinary skillin the art can also determine the rate of hydrolysis of thepolyphosphazenes of diverse structures as described herein, and will beable to select the polyphosphazene that provides the desiredbiodegradation profile for the targeted use.

The term biodegradable polymer refers to a polymer that degrades withina period that is acceptable in the desired application, less than weeksor months, when exposed to a physiological solution of pH between 6 and8 having a temperature of between about 25° C. and 37° C.

Polyphosphazenes can be made by displacing the chlorines inpoly(dichlorophosphazene) with a selected substituent group or groups.Desired proportions of hydrolyzable to nonhydrolyzable side chains inthe polymer can be achieved by adjusting the quantity of thecorresponding nucleophiles that are reacted withpoly(dichlorophosphazene). The preferred polyphosphazenes have amolecular weight of over 1,000.

Methods for synthesis of polyphosphazenes are described by Allcock, H.R.; et al., Inorg. Chem. 11, 2584 (1972); Allcock, et al.,Macromolecules 16, 715 (1983); Allcock, et al., Macromolecules 19, 1508(1986); Allcock, H. R.; Gebura, M.; Kwon, S.; Neenan, T. X.Biomaterials, 19, 500 (1988); Allcock, et al., Macromolecules 21, 1980(1988); Allcock, et al., Inorg. Chem. 21(2), 515-521 (1982); Allcock, etal., Macromolecules 22, 75 (1989); U.S. Pat. Nos. 4,440,921, 4,495,174and 4,880,622 to Allcock, et al.; U.S. Pat. No. 4,946,938 to Magill, etal., and Grolleman, et al., J. Controlled Release 3, 143 (1986), theteachings of which are specifically incorporated herein. The synthesisof ionically crosslinkable poly(carboxylatophenoxy)phosphazene, and thepreparation of hydrogels from this polymer, is taught in U.S. Pat. No.5,053,451. Other patents on poly(organophsphazenes) include U.S. Pat.Nos. 4,440,921, 4,880,622, 3,893,980, 4,990,336, 4,975,280, 5,104,947,and 4,592,755.

Most preferably, the polyphosphazene is a high molecular weight,water-soluble anionic polyphosphazene, in which a majority of sidegroups in phosphazene polyelectrolytes are ionic. Carboxylic acid groupsare an example of preferred ionic groups.

One example of a preferred polyanionic phosphazene ispoly(carboxylato-phenoxy)phosphazene (PCPP).

2. Other Water Soluble Polymers With Charged Side Groups

A wide variety of water soluble or dispersible polymers with ionic sidegroups are known or can be easily designed by one of ordinary skill inthe art of polymer synthesis. The polymers are in general those that arebiocompatible, optionally biodegradable, and have acidic or basicsubstituent groups as described in detail above. The polymers caninclude non-ionic monomers that impart desired properties to thepolymer. The polymer can be a condensation polymer or addition polymer.Nonlimiting examples of monomers that can be included in condensationpolymers are hydroxyacids such as lactic acid, glycolic acid, andhydroxybutyric acid, and dicarboxylic acids. Examples of useful polymersincludes poly(acrylic acid) (PAA); poly(methacrylic acid) (PMA); andmethacrylic acid copolymers such as Eudragit L100 and S100.Biodegradable polymers include those that degrade enzymatically andthose that degrade hydrolytically.

Methods for synthesizing the other polymers described above are known tothose skilled in the art. See, for example Concise Encyclopedia ofPolymer Science and Polymeric Amines and Ammonium Salts, E. Goethals,editor (Pergamen Press, Elmsford, N.Y. 1980). Many, such as poly(acrylicacid), are commercially available.

One preferred polymer is poly(meth)acrylic acid (wherein the term(meth)acrylic refers to either polymethacrylic acid or polyacrylic acid)or a copolymer of methacrylic acid or acrylic acid with anotherunsaturated monomer that can be ionic or non-ionic. Pharmaceuticalapplications of poly(meth)acrylic acids are well known [K. O. R. Lehmanin "Aqueous polymeric coatings for pharmaceutical dosages forms" Ed. J.W. McGinity, Marcel Dekker, 1989, pp. 1-93]. Because of its excellentbiocompatibility, poly(meth)acrylic acid and copolymers of (meth)acrylicacid are used for artificial implants, dental prosthesis, contactlenses, ointments and coatings for gastroresistant-enterosolubleformulations.

Examples of anionic poly(meth)acrylic acids include but are not limitedto copolymers of methacrylic acid and ethyl acrylate-poly[(methacrylicacid)-co-(ethyl acrylate)], also known as Eudragit. The chemicalcompositions of commercially available polymers (Eudragit L and S) areshown below. ##STR1##

The ratio of the free carboxyl groups to the ester groups isapproximately 1:1 in Eudragit L and approximately 1:2 in Eudragit S. Themean molecular weight is calculated from viscosity measurement and equalto 135,000 Da. The polymers correspond to USP/NF, "Methacrylic AcidCopolymer Type A" (Eudragit , L) or "Type B" (Eudragit S). Thecopolymers are practically insoluble in water, but soluble in 1N sodiumhydroxide solution (upon neutralization of carboxyl groups) to giveclear to slightly opalescent solutions.

B. Crosslinking the-Polymers With Multivalent Ions to Form a Hydrogel

The water-soluble polymer with charged side groups is crosslinked byreacting the polymer with an aqueous solution containing multivalentions of the opposite charge, either multivalent cations if the polymerhas acidic side groups or multivalent anions if the polymer has basicside groups.

The term "pharmaceutically acceptable cation" refers to an organic orinorganic moiety that carries a positive charge that can be administeredin vivo without undue toxicity to the host.

The term polyelectrolyte, as used herein, refers to a polymer with ionicside groups.

1. Cross-linking the Polymers With Acidic Side Groups by MultivalentCations

The preferred cations for cross-linking the polymers with acidic sidegroups to form a hydrogel are divalent and trivalent cations such ascopper, calcium, aluminum, magnesium, strontium, barium, tin, chromium,and preferably zinc, although di-, tri- or tetra-functional organiccations such as salts of nitrogenous bases, for example, alkylammoniumsalts, such as piperidine dihydrochloride, the salt of ethylene diaminetetra(acetic acid), can also be used. Aqueous solutions of the salts ofthese cations are added to the polymers to form soft, highly swollenhydrogels and membranes. The higher the concentration of cation, or thehigher the valence, the greater the degree of polymer cross-linking.Concentrations as low as 0.005M have been demonstrated to crosslink thepolymer. Higher concentrations are limited by the solubility of thesalt.

2. Cross-Linking the Polymers With Basic Side Groups With MultivalentAnions

The preferred anions for cross-linking the polymers to form a hydrogelare divalent and trivalent anions such as low molecular weightdicarboxylic acids, for example, terepthalic acid, sulfate ions andcarbonate ions. Aqueous solutions of the salts of these anions are addedto the polymers to form soft, highly swollen hydrogels and membranes, asdescribed with respect to cations.

C. Crosslinking the Polymers With Multivalent Polyions to Form aSemi-Permeable Membrane

In some embodiments, additional surface groups on the hydrogel polymerare reacted with polyions of opposite charge to form a semi-permeablemembrane on the surface of the hydrogel. The complexed polymer is stableand forms a semipermeable membrane on the microcapsules. Thepermeability of this membrane for a given entity depends on themolecular weight of the polyion. When the hydrogel is in the form of amicrocapsule (or microsphere), the core hydrogel can then be liquifiedby removing the multivalent ions, for example, by dialysis or additionof a chelating agent.

1. Multivalent Polycations Useful for Crosslinking

A variety of polycations can be used to complex and thereby stabilizethe polymer hydrogel into a semi-permeable surface membrane. Examples ofmaterials that can be used include polymers having basic reactive groupssuch as amine or imine groups, having a preferred molecular weightbetween 3,000 and 100,000, such as polyethylenimine and polylysine.These are commercially available. A preferred polycation ispoly(L-lysine). Examples of synthetic polyamines include but are notlimited to polyethyleneimine, poly(vinylamine), and poly(allyl amine).There are also natural polycations, such as the polysaccharide chitosan,but these are not preferred.

2. Multivalent Polyanions Useful for Crosslinking Polymers With BasicSide Groups

Polyanions that can form a semi-permeable membrane by reacting withbasic surface groups on the polymer hydrogel include polymers andcopolymers of acrylic acid, methacrylic acid, and other derivatives ofacrylic acid, polymers with pendant SO₃ H groups such as sulfonatedpolystyrene, and polystyrene with carboxylic acid groups.

D. Modification of Acidic Groups on the Polymer Backbone

1. Coupling of Molecules to Modify the Surface or to Target theMicroparticle

The ionically crosslinkable groups on the polymer can be modified bycovalently coupling a poly(alkylene glycol) such as poly(ethyleneglycol), proteins, peptides, oligosaccharides, carbohydrate, lipids,nucleotide sequences or other molecules to target the microparticles tospecific regions of the body or to certain cell types, or minimizetissue adhesion or uptake by the reticuloendothelial system (RES). Thetargeting molecule can be, for example, a protein or peptide such as ahormone, antibody or antibody fragment such as the Fab or Fab₂ antibodyfragments, or a specific cell surface receptor ligand that localizes atthe target material.

2. Methods for Coupling Molecules to the Microparticles

The coupling involves forming ester, thioester, amide, or sulfamidelinkages. Coupling hydroxy, thio, or amine groups with carboxy orsulfoxy groups is known to those skilled in the art.

The polymers can contain various functional groups, such as hydroxy,thio, and amine groups, that can react with a carboxylic acid orcarboxylic acid derivative under the coupling conditions. Reactivefunctional groups not involved in the coupling chemistry must beprotected to avoid unwanted side reactions. After the carboxylic acid orderivative reacts with a hydroxy, thio, or amine group to form an ester,thioester, or amide group, any protected functional groups can bedeprotected by means known to those skilled in the art.

The term "protecting group" as used herein refers to a moiety whichblocks a functional group from reaction, and which is cleavable whenthere is no longer a need to protect the functional group. Suitableprotecting groups for the hydroxyl group include, but are not limitedto, certain ethers, esters and carbonates (Greene, T. W. and Wuts, P. G.M., "Protective groups in organic synthesis," John Wiley, New York, 2ndEd. (1991)). Suitable protecting groups for the carboxyl group include,but are not limited to, those described in Green and Wuts, ProtectingGroups in Organic Synthesis, John Wiley (1991). Side-chainfunctionalities such as carboxylic acids, alcohols, and amines mayinterfere with the coupling chemistry and must be appropriatelyprotected.

As used herein, "side-chain functionality" refers to functional groups,such as hydroxy, thio, amine, keto, carboxy, alkenyl, alkynyl, carbonyl,and phosphorus derivatives such as phosphate, phosphonate andphosphinate in the polymer or material to be covalently attached to thepolymer, that is not involved in coupling to form an ester, thioester,amide or sulfamide bond. Examples of suitable protecting groups are wellknown to those skilled in the art. See, generally, Greene and Wuts,Protecting Groups in Organic Chemistry, John Wiley (1991).

Examples of protecting groups for amine groups include, but are notlimited to, t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz),o-nitrobenzyloxycarbonyl, and trifluoroacetamide (TFA).

3. Coupling Other Groups on the Polymer with Biological Materials

Amine groups on a polymer backbone can be coupled with amine groups on apeptide by forming a Schiff base, using coupling agents such asglutaraldehyde. An example of this coupling is described by Allcock, etal., Macromolecules, Vol. 19(6), pp. 196 (1986), hereby incorporated byreference. In this example, trypsin was bound to amine groups on apolyphosphazene. An aminophenoxy polyphosphazene and trypsin were addedto a buffer solution. Glutaric dialdehyde was added to the solution, andthe solution was kept at 0° C. for 20 hours.

The amount of bound trypsin was determined by washing the excess trypsinfrom the polymer, using Lowry protein measurement to determine theamount of unbound trypsin, and calculating the amount of bound trypsinby difference.

Amine groups can also be coupled with DCC or other dehydrating agents,as described above, with carboxy groups on amino acids, proteins orpeptides.

Alternatively, one can incorporate amino acids, proteins, or peptidesinto the polymer backbone by displacing chlorines on chlorine-containingpolyphosphazenes, such as polydichlorophosphazene. Since the carboxylatesalt of carboxy groups can also displace the chlorine, the carboxygroups must be protected. Examples of this chemistry are described byAlcock, et al., Macromolecules, Vol. 10(4), pp. 824-831 (1977), herebyincorporated by reference.

Additionally, amine groups can be converted to diazonium salts, whichcan be reacted with amine or hydroxy groups on biological materials. Anexample of this coupling is described by Allcock, et al.,Macromolecules, Vol. 16(9), pp. 1405 (1983), hereby incorporated byreference.

For example, poly(15% 4-aminophenoxy/85% phenoxy) phosphazene isdissolved in THF containing HCl, and the solution cooled to 0° C. Asolution of NaNO₂ is added to form the diazonium salt. A bufferedsolution of d,l-epinephrine is added, and the reaction proceeded in thedark, at 0° C., for 14 hours. A 60% yield is obtained.

Phenol or alcohol substituents on the polymer can be coupled withcarboxylic or sulfonic acid groups on biological materials, such as acarboxy group on an amino acid, protein or peptide. The conditions forthese coupling reactions are described above.

Aldehyde groups on polymers can be coupled with amines, as describedabove, by forming a schiff base. An example of this coupling isdescribed by Allcock and Austin, Macromolecules, Vol. 14, pp. 1616(1981), hereby incorporated by reference.

For example, Hexakis(4-aminophenoxy) cyclotriphosphazene (1 gram, 1.2mmol) is dissolved in diethylene glycol (35 mL), and 16 mmol citral isadded. The mixture is stirred at 25° C. for 2 hours, HCl (18 mmol) isadded, and the solution is warmed. Additional citral (15 mmol) is added.After 15 minutes at room temperature, 10 mL of water are added. Afterworkup and recrystallization, the resulting yield is 88%.

II. Methods of Making Microparticles

The method of preparing the microparticles should be selected to providea microparticle having the desired size for the intended use. In apreferred embodiment for the preparation of injectable microparticlescapable of passing through the pulmonary capillary bed, themicroparticles should have a diameter of between approximately one andseven microns. Larger microparticles may clog the pulmonary bed, andsmaller microparticles may not provide sufficient echogenicity. Largermicroparticles may be useful for administration routes other thaninjection, for example oral (for evaluation of the gastrointestinaltract) or by inhalation.

A. Preparation of a Polymer Solution

In general, the polymer is dissolved or dispersed into a solution whichis then sprayed into a solution of crosslinking counterions. This istypically an aqueous solution or dispersion that can includewater-miscible organic solvents, including but not limited to dialkylsulfoxides, such as dimethyl sulfoxide DMSO); dialkyl formamides, suchas dimethyl formamide (DMF); C₁₋₅ alcohols, such as methanol andethanol; ketones such as acetone and methyl ethyl ketone; and etherssuch as tetrahydrofuran (THF), dibutyl ether and diethyl ether. Thesolution can be neutral, acidic or basic, and can contain salts orbuffers. If the ionic polymer is insoluble in water, or insufficientlydispersible, the polymer can be converted to its conjugate acid or basethat is typically more water soluble, and that conjugate acid or basethen exposed to the di- or multivalent counterion for crosslinking.

B. Gases to be Encapsulated

The ratio of polymer to gas is determined based on the gas that is to beencapsulated, for example, as required to produce a particle size smallenough to be injected. Any desired inert gas can be incorporated intothe polymeric materials at the time of hydrogel formation, includingair, argon, nitrogen, carbon dioxide, nitrogen dioxide, methane, helium,neon, oxygen and perfluorocarbon. Sterilized air or oxygen is apreferred imaging contrast agent.

C. Atomization of Polymer Solution Into a Crosslinking Solution

There are at least two methods for the preparation of ingectablemicroparticles. In one method, a jet head is used that allows theco-extrusion of a solution of polymer and air to produce nascentmicroencapsulated air bubbles which fall into a hardening solution ofcounterions. A second method employs ultrasound to introducecavitation-induced bubbles into the polymer before capsule formation byspraying. To incorporate gases other than air, a solution of the desiredpolymer is placed in an atmosphere of the desired gas and sonicated fora sufficient amount of time before crosslinking to ensure that gasbubbles are dispersed throughout the microparticulates. In either case,the determining factors on size of resulting microparticles will be theselection and concentration of polymer and solvent, and size of dropletsformed by the atomizer.

1. Preparation of One to Ten Micron Microparticles

An example of an air-atomizing device is a Turbotak, from Turbotak,Inc., Waterloo, Ontario. A Turbotak is a hollow stainless steelcylinder, 2.64 cm. wide × 4 cm. long. Liquid is fed into the Turbotakfrom the top and pressurized air is fed from the side. The pressurizedair mixes with the liquid, forcing tiny liquid droplets out through theorifice of the nozzle. The air pressure can be set to between 50 and 80psig. The distance between the orifice of the Turbotak and the pancontaining the crosslinking ions is fixed at between about one to twofeet. The size of the nozzle orifice is 1 to 2 mm in diameter.

Air can be pressurized with a syringe pump such as a Razel pump, havinga flow rate in the range of between 5 ml/hr and 30 ml/hr or a Sage pump,having a flow rate in the range of between 0.02 ml/min and 126 ml/min.

Mixing pressurized air with a polymer solution aerates the polymersolution and produces a high yield of air-encapsulated polymericmicroparticles. Even without sonicating the polymer solution,microparticles produced using the Turbotak nozzle have entrapped air, asseen by light microscopy.

2. Method for the Preparation of Larger Microparticles

Larger microparticles can be prepared using a droplet-forming apparatusby spraying an aqueous solution of polymer containing the agent ofinterest through an apparatus such as a plastic syringe, where thepolymer solution is extruded through a needle located inside a tubethrough which air flows at a controlled rate.

The rate of polymer extrusion is controlled, for example, by a syringepump. Droplets forming at the needle tip are forced off by the coaxialair stream and collected in the crosslinking solution, usually anaqueous solution of bi- or trivalent ions, where they cross-link and arehardened, for example, for between 15 and 30 minutes.

The shape and size of these microparticles depend on the polymer andcross-linker concentrations and parameters such as the polymer extrusionrate, air flow, and needle diameters used in the microencapsulationprocedure.

A typical example for microparticle preparation utilizes PCPP polymerconcentrations of between 1 and 5% (w/v), preferably around 2.5%, andcalcium chloride concentrations of between 3 and 7.5% (w/v), preferably7.5%, respectively. Polymer extrusion rates are between 50 and 100mL/hour, preferably 70 mL/hour. Air flow rates are in the range of 5L/hour. Needle diameters of between 18 and 26 gauge (G), preferablyaround 20 gauge, are used. Using the preferred conditions, the resultantmicroparticles are spherical with diameters in the range of 400-700microns. In general, microparticles as small as 30 μM can be preparedusing this technique.

Macrospheres with millimeter diameters can be prepared by extruding thepolymer through pasteur pipets or their equivalent.

D. Incorporation of Contrast Agents

Other contrast agents can be incorporated in place of the gas, or incombination with gas, using the same methods. These are useful inimaging using the more common techniques such as ultrasound, magneticresonance imaging (MRI), computer tomography (CT), x-ray, as well as theless common positron emission tomography (PET) and single photonemission computerized tomography (PET).

Examples of suitable materials for ultrasound include the gasesdiscussed above.

Examples of suitable materials for MRI include the gatalinium chelatescurrently available, such as diethylene triamine pentacetic acid (DTPA)and Gatopentotate dimeglumine, as well as iron, magnesium, manganese,copper and chromium. These are typically administered in a dosageequivalent to 14 ml for a 70 kg person of a 0.5 M/liter solution.

Examples of materials useful for CT and x-rays include iodine basedmaterials for intravenous administration such as ionic monomers typifiedby Diatrizoate and iothalamate (administered at a dosage of 2.2 ml of a30 mg/ml solution), non-ionic monomers typified by iopamidol, isohexol,and ioversol (administered at a dosage of 2.2 ml 150-300 mg/ml),non-ionic dimers typified by iotrol and iodixanol, and ionic dimers, forexample, ioxagalte. Other useful materials include barium for oral use.

E. Processing the Polymeric Microparticles to Liquify the Core

The polyionic-coated hydrogel microparticles are collected and furthertreated with buffer to remove the uncomplexed multivalent ions. Forexample, to remove uncomplexed multivalent cations, microparticles canbe treated with 0.9% (w/v) KCl with the pH adjusted to around 8.0. TheKCl solution dissolves the internal gel, without affecting the externalmembrane. Other methods can also be used to liquefy the internal gel,including using chelators such as EDTA and sodium citrate.

III. Detecting Contrast Agent-Encapsulating Microparticles

1. Detection of Gas Microbubbles

The relatively homogenous population of gel microparticles, filled withcontrast agents, can be seen by an inverted microscope. Mostmicroparticles produced by the first method are smaller than sevenmicrons in diameter. Particle size analysis can be performed on aCoulter counter.

Due to their in vivo stability their potential application is extendedbeyond vascular imaging to liver and renal diseases, fallopian tubediseases, detecting and characterizing tumor masses and tissues, andmeasuring peripheral blood velocity. The microparticles can optionallybe linked with ligands that minimize tissue adhesion or that target themicroparticles to specific regions.

The method for imaging by detection of gas bubbles in a patient uses atransducer which produces pulses, illustrative of ultrasonic acousticenergy, having predetermined frequency characteristics. A first pulsehas an increasing frequency with time, and a second pulse has adecreasing frequency with time. Imaging arrangements produce images ofthe region within the specimen after exposure to the first and secondpulses.

The conventional technique for determining the presence of bubbles inthe blood stream uses a Doppler shift in the frequency of the ultrasonicacoustic energy which is reflected by the blood. The amplitude of theDoppler bubble signal increases nearly proportionally with increases inthe radius of the bubble. The human hearing mechanism is considered themost accurate processor for recognizing whether bubble signals arepresent or absent. For this reason, it is preferable to have a skilledoperator to obtain satisfactory results using Doppler blood flowmonitoring equipment.

To determine whether the air-filled microparticles are useful for invivo imaging, the following in vitro method, described in more detail inthe following examples, can be used.

Microparticles prepared by the above methods are suspended in a cappedtissue culture tube. For ultrasound imaging, the tubes are placed on topof a pad covered with coupling medium above the transducer. Thetransducer is held in place at roughly a 90° angle of incidence tominimize any motion artifacts. The transducer acts as a transmitter andalso receives ultrasound radiation scattered back from the tube. B-modeand Doppler images are established for tubes filled with polymericmicroparticles and the resulting images are compared with a controlconsisting of an image from a tube containing buffer alone. The B-modeof display gives a two dimensional image of a slice through the scannedtube.

This method was used to obtain in vitro results on the microparticles inthe working examples described below. These results correlated well withthe in vivo results, as shown by Doppler imaging techniques (describedbelow). Since the in vitro and in vivo data showed a high degree ofcorrelation in the working examples, this test is reasonably predictiveof the in vivo stability of microparticles.

2.Detection of Other Contrast Agents

Other means of detection include PET (positron emission tomograph),(CAT) computer assisted tomography, x-rays, fluoroscopy, and MRI(magnetic resonance imaging). These are conducted using the standardtechniques and equipment as used with other commercialy availablecontrast agents.

The methods and compositions described above will be further understoodwith reference to the following non-limiting examples.

EXAMPLE 1 Preparation of One to Ten Micron Eudragit S 100 Microparticles

The microencapsulation procedure, optimized to produce microparticles inthe size range of 1-10 μm, is as follows:

Preparation of Eudragit S 100 (5.4 w/v) Solution

1 gram of S 100 was dissolved overnight, at room temperature, withstirring in 1N KOH, such that the carboxylic acid groups wereneutralized. This required adding approximately 190 mg of KOH per gramof S 100. The solution was then diluted with double-distilled water togive a final polymer concentration of 5% (w/v) and solution pH around7.0 (due to polymer neutralization). If the pH was greater than 7.0, thepH was adjusted with 1N HCl.

Preparation of S 100 Microparticles

A solution of Eudragit S 100 (5% w/v) containing 0.2% Tween 20 wassonicated with a horn sonicator (output 8, for 15 minutes, in anice-bath) to produce a gassed S 100 solution that was highly aerated andstable for hours. The gassed solution was extruded from a syringe pump(Razel Instrument) at 150 μl min into an air-atomizing device (Turbotak,Turbotak, Inc., Waterloo, Ontario) and sprayed into a pan containing 250mL of 15% calcium chloride solution with 0.5% Tween 20.

Upon contact with calcium chloride solution, S 100 was cross-linked bythe divalent calcium ions to produce a relatively homogenous populationof spherical gel microparticle, filled with air bubbles. The presence ofair bubbles was shown by looking at the microparticle through aninverted microscope. Most S100 microparticles were smaller than 7 μm.The yield of microparticle after one passage through a 7 μm spectrumfilter (polyester-based filter, Spectrum) was more than 90%. Particlesize analysis (Coulter counter) gave the following number of particles:90% of the particles were smaller than 5.448 μm, 75% were smaller than3.763 μm, 50% were smaller than 2.692 μm, 25% were smaller than 2.058μm, and 10% were smaller than 1.715 μm. Analysis also indicated that 25%of total particle volume belonged to particles with diameter less than7.65 μm.

EXAMPLE 2 Preparation of PCPP Microparticles

Preparation of PCPP (2.5% w/v)

100 mg of PCPP were dissolved in 1 mL 30 mg/mL Na₂ CO₃ (overnight, atroom temperature, stirring) and then diluted with phosphate-bufferedsaline (PBS), pH 7.4 to give a final polymer concentration of 2.5% (w/v)and solution pH of 7.4 (due to polymer deprotonation), if not, the pHwas adjusted with 1N HCl.

Preparation of PCPP Microparticles

A solution of PCPP (2.5% w/v) containing 0.2% Tween 20 was sonicatedwith a horn sonicator (output 8, for 5 minutes, in an ice-bath) toproduce a gassed PCPP solution that was highly aerated and stable forhours. The gassed solution was extruded from a syringe pump (RazelInstrument) at 150 μl/min into an air-atomizing device (Turbotak,Turbotak, Inc., Waterloo, Ontario) and sprayed into a pan containing 250mL of 7.5% calcium chloride solution containing 0.5% Tween 20. Uponcontact with calcium chloride solution, PCPP was crosslinked by thedivalent calcium ions to produce a relatively homogenous population ofspherical gel microparticle, filled with air bubbles. The presence ofair bubbles in the microparticle was shown by looking at themicroparticle through an inverted microscope. Particle size analysis(Coulter counter) gave the following number of particles: 90% of theparticles were smaller than 14.77 μm, 75% were smaller than 9.253 μm,50% were smaller than 5.84 μm, 25% were smaller than 4.166 μm, and 10%were smaller than 3.372 μm. Analysis also indicated that 10% of totalparticle volume belonged to particles with diameter less than 14.57 μm.

EXAMPLE 3 Preparation of Poly(methylmethacrylate) MicroparticlesCrosslinked With Zinc

A solution of Eudragit™ S100 (5% w/v) containing 0.25% Tween™ 20 issonicated with a horn sonicator (output 8, for 5 minutes, in an icebath), or with a vortex mixer (5 minutes) to produce a gassed S100solution that is highly aerated and stable for hours. The gassedsolution is extruded from a syringe pump (Razel Instrument) at 500μl/min into an air-atomizing device (Turbotak, Turbotak, Inc.,Waterlook, Ontario) and sprayed into a pan containing 250 ml of 0.5%zinc chloride solution containing 0.5% Tween™ 20. The air pressure isset to 12 psi. The distnace between the orifice of the Turbotak and thepan is fixed at 16 cm. Even without sonication of polymer solution,microparticulates produced by the Turbotak nozzle have entrapped air, asseen by light microscopy.

Upon contact with the zinc chloride solution, S100 is cross-linked bythe divalent cations to produce a relatively homogeneous population ofspherical microparticulates, filled with air bubbles, as seen with aninverted microscope. All S100 microparticulates were 4.5 microns indiameter, with nearly zero standard deviation, by light scattering.

Crosslinking with zinc is advantageous since it is expected that theseions will have less of a physiological effect than calcium. Moreover,the zinc ions produced an extremely narrow size distribution, with noseiving or filtration required, and the resulting microparticulates werehighly echogenic.

EXAMPLE 4 In vitro Ultrasound Imaging

Air-filled microparticles, at a concentration of 1×10⁶ particles/mL,were suspended in a capped tissue culture tube with a length of 15 cm.and a depth of 1.5 cm. For ultrasound imaging, the tubes were placed ontop of a pad containing a transducer, with coupling medium above thetransducer. The transducer was held in place at a 90° angle of incidenceto minimize any motion artifacts. B-mode and Doppler images wereestablished for a tube filled with polymeric microparticles and theresulting images were compared with a control consisting of an imagefrom a tube that contained buffer alone.

A comparison was made between the B-mode sector images of tubes filledwith saline and tubes filled with S 100 microparticles (3-5 μm indiameter) and with PCPP microparticles (3-5 μm in diameter),respectively.

The brightness seen at the bottom of the picture of the tube filled withsaline reflects the saline/air interface within the tube. No echo wasreturned from the inside of the tube. In the tubes filled with bothtypes of polymeric microparticles, a very strong echo was returned tothe transducer, giving a clear image of the tube, and demonstrating thatthe polymeric microparticles were highly echogenic. The polymericmicroparticles were still highly echogenic, 24 hours and a week afterpreparation (during which they were kept at room temperature), asverified by subsequent B-mode images.

EXAMPLE 5 In Vivo Ultrasound Imaging

In vivo imaging was performed with white rabbits weighing approximately3.5 kg. The rabbits were anesthetized with 1 mL/kg Rabbit Mix (8:5:2ratio of xylazine hydrochloride, 20 mg/mL; ketamine hydrochloride, 10mg/mL; and acepromazine maleate, 100 mg/mL) administeredintramuscularly.

Intravenous injections of saline or air-filled polymeric microparticleswere performed through the marginal ear vein using a 23-gauge butterflycatheter. Ultrasound imaging was performed with the Acuson scanner usinga 7.5-MHz high-resolution linear transducer. Imaging of the aorta wasperformed before and after administering the contrast agent. Both B-modeand Doppler images were established.

The color Doppler ultrasound is a particularly sensitive detector ofbubbles. The effect of an ultrasonic contrast agent is readilyidentifiable by a sudden marked increase in the amplitude of the audibleDoppler signal and easily recognized by all listeners. There is also achange in the quality of the Doppler sound, consisting primarily of anapparent increase in pitch.

To verify that the air bubbles remained encapsulated in themicroparticle during in vivo application, a B-mode sector image of anaorta was taken after first injecting 5 mL saline, and then injecting 1mL and then 2 mL of solutions containing PCPP polymeric microparticles(1×10⁶ particles/mL) with a period of 5 minutes between each injection.No echoes were reflected from the aorta after the saline injection.However, immediately after injecting polymeric microparticles, the aortawas filled with echogenic microparticles. Injecting an additional 2 mLof microparticles resulted in a very strong echo, giving a clear imageof the blood vessel and demonstrating that these microparticles werehighly echogenic. The echo from the aorta lasted for more than 15minutes, and it seemed that its intensity did not decline with time.

A color Doppler image of the aorta approximately 15 minutes afterinjection showed a significant increase in the image and the signal.Pictures were taken 15 and 20 minutes after injection, demonstratingthat the PCPP microparticles are very stable in vivo (can survive thehigh pressure of the left chambers of the heart), can pass the capillarybed of the lung and are very echogenic.

Variations and modifications of the compositions, methods of preparingthe compositions, and methods of using the compositions will be obviousto those with ordinary skill in the art. It is intended that all ofthese variations and modifications be included within the scope of theappended claims.

We claim:
 1. A method for imaging a predetermined area of a mammal inmedical procedures, the method comprising the steps of:a) providingmicroparticles formed by dispersing or dissolving an imaging contrastagent in an aqueous solution or dispersion of an ionically crosslinkablesynthetic polymer that has charged side groups, wherein the polymer isselected from the group consisting of poly(phosphazenes), poly(acrylicacids), poly(methacrylic acids), copolymers of acrylic acid andmethacrylic acid, copolymers of acrylic acid or methacrylic acid andpolyvinyl ethers or poly(vinyl acetate), and sulfonated polystyrene, andreacting the polymer with a multivalent ion of the opposite charge toform synthetic microparticles comprising a hydrogel having the imagingcontrast agent incorporated therein in an amount effective to bedetectable by an imaging technique after administration to a patient, b)administering the microparticles to a mammal; and c) scanning apredetermined area of the mammal to obtain an image of the area.
 2. Themethod of claim 1 wherein the diameter of the microparticles is betweenone and ten microns.
 3. The method of claim 1 wherein the polymer issoluble prior to crosslinking in an aqueous solution selected from thegroup consisting of water, aqueous alcohol, and buffered aqueous saltsolutions.
 4. The method of claim 1 wherein the charged groups on thesurface of the hydrogel are further complexed with a multivalent polyionof the opposite charge to form a semi-permeable membrane.
 5. The methodof claim 4 wherein the hydrogel is liquefied within the semi-permeablemembrane by removing the multivalent ions.
 6. The method of claim 1wherein the multivalent ion is selected from the group consisting ofcalcium, copper, aluminum, magnesium, stromium, barium, tin, chromium,zinc, organic cations, dicarboxylic acids, sulfate ions and carbonateions.
 7. The method of claim 1 wherein the imaging contrast agent isgas, and the gas is entrappeal in the polymer solution or dispersion bysonication of the polymer in the presence of the gas in step (a).
 8. Themethod of claim 1 wherein the imaging contrast agent is gas and the gasis mixed with the polymer solution or dispersion and atomized through anorifice forming droplets which are dispersed into a crosslinkingsolution comprising the multivalent ion in step (a).
 9. The method ofclaim 1 wherein the contrast agent is selected from the group consistingof air, argon, nitrogen, carbon dioxide, nitrogen dioxide, methane,helium, neon, oxygen, perfluorocarbon, a gatalinium chelate, iron,magnesium, manganese, copper, chromium, a radiolabeled compound, aniodine-containing material and barium.
 10. The method of claim 1 whereinstep c) is conducted using an imaging technique selected from the groupconsisting of ultrasound, magnetic resonance imaging, computertomography, x-ray, positron emission tomography and single photonemission computerized tomography.
 11. The method of claim 10 whereinstep c) is conducted using ultrasound imaging techniques.
 12. The methodof claim 11 wherein the contrast agent is selected from the groupconsisting of air, argon, nitrogen, carbon dioxide, nitrogen dioxide,methane, helium, neon, oxygen and perfluorocarbon.
 13. The method ofclaim 1 wherein the microparticles are administered by an administrationroute selected from the group consisting of injection, oraladministration, and inhalation.
 14. The method of claim 9 wherein theiodine-containing material is selected from group consisting ofdiatrizoate, iothalamate, iopamidol, isohexol, ioversol, iotrol,iodixanol and ioxagalte.